Enzymes for the production of 2-keto-L-gulonic acid

ABSTRACT

Mutants of 2,5-diketo-D-gluconic acid reductase A, an enzyme used to produce 2-keto-L-gulonic acid, a precursor of ascorbic acid (vitamin C) are prepared by site-directed mutagenesis. These mutants have increased catalytic activity, increased expression levels, and/or enhanced temperature stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.07/941,414 (filed on Sep. 8, 1992) now U.S. Pat. No. 5,376,544 Dec. 27,1994.

FIELD OF THE INVENTION

The present invention relates to improved mutant forms of anindustrially valuable enzyme as a result of site-directed mutagenesis.More specifically, the invention relates to mutated forms of2,5-diketo-D-gluconic acid (2,5-DKG) reductase A, a naturally occurringvariant of DKG reductase. The mutated forms show improved catalyticactivity for converting 2,5-DKG stereoselectively into 2-keto-L-gulonicacid (2-KLG), a precursor of ascorbic acid (vitamin C). In addition, themutated forms have increased in-vivo expression levels and/or improvedtemperature stability.

BACKGROUND OF THE INVENTION

Due to the expanding health consciousness of people around the world,there has been an increasing demand for vitamin C. Also contributing tothe demand for ascorbic acid is its widespread use as an antioxidant forpreserving food. One approach for satisfying this demand is to achieveincreased production of 2-KLG, an intermediate in the production ofascorbic acid. The intermediate, 2-KLG, can be easily converted toascorbic acid through acid or base catalyzed cyclization. It also has agreater stability and shelf life than ascorbic acid. Therefore, ratherthan producing ascorbic acid directly, it is more practical to stockpile2-KLG for subsequent conversion to ascorbic acid.

A number of species of a first group of microorganisms, Erwinia,Acetobacter, and Gluconobacter, can produce 2,5-DKG from D-glucose. Asecond group of microorganisms from the coryneform group of bacteria(Corynebacterium, Brevibacterium, and Arthrobacter) as well as speciesof Micrococcus, Staphylococcus, Pseudomonas, Bacillus, and Citrobacterare capable of converting 2,5-DKG, produced by a microorganism of thefirst group, to 2-KLG. This cofermentation of appropriate microorganismsto produce 2-KLG was simplified by combining the relevant traits of boththe Erwinia sp. and the Corynebacterium sp. in a single microorganism(Anderson et al., Science 23: 144-149 (1985)). This was accomplished byidentifying the 2,5-DKG reductase in the Corynebacterium sp. thatconverts 2,5-DKG into 2-KLG. The gene for this reductase was then clonedand expressed in Erwinia herbicola, a bacterium of the familyEnterobacteriaceae that converts D-glucose into 2,5-DKG in a singlefermentation. The resulting recombinant bacterial strain, with 2,5-DKGreductase as the pivotal enzyme, was able to convert D-glucose into2-KLG in a single-fermentation process (Lazarus et al. Fourth ASM Conf.Genet. Molec. Biol. Indust. Microorg., 187-193 (1989)).

Improving the catalytic efficiency of 2,5-DKG reductase, in thesingle-fermentation process, is a significant way to increase theproduction of 2-KLG. Also, a purified 2,5-DKG reductase A with increasedcatalytic activity could be used in an in vitro process for theconversion of 2,5-DKG to 2-KLG. For example, such a process would permitcontinuous production of 2-KLG through immobilization of the purifiedenzyme on a solid support.

According to the Michaelis-Menten scheme set out below, the ##STR1##efficiency of an enzymatic reaction can be measured by two kineticparameters, kcat and Km. The catalytic rate constant, kcat, also knownas the turnover number, is a measure of the breakdown of theenzyme-substrate (ES) complex. It also represents the maximum number ofsubstrate molecules (S) converted to product (P) via an ES complex peractive site of the enzyme (E) per unit time. Vmax is the maximalvelocity or rate of the enzyme catalyzed reaction when the enzyme issaturated with substrate. Therefore, Vmax is constant at saturatingsubstrate concentration and remains unchanged with any increase insubstrate concentration. The kcat at saturating substrate concentrationsis related to Vmax and the total enzyme concentration, [E_(T) ], by thefollowing equation: Vmax=kcat [E_(T) ]. The Michaelis constant, Km, isequal to the dissociation constant of the ES complex. Therefore, Km is ameasure of the strength of the ES complex. In a comparison of Km's, alower Km represents a complex with a stronger, more favorable binding,while a higher Km represents a complex with a weaker, less favorablebinding. The ratio, kcat/Km, called the specificity constant, representsthe specificity of an enzyme for a substrate, i.e., the catalyticefficiency per enzyme molecule for a substrate. The larger thespecificity constant, the more preferred the substrate is by the enzyme.

Impressive yields of 2-KLG have been achieved with a Corynebacterium2,5-DKG reductase (2,5-DKG reductase A, also known as 2,5-DKG reductaseII) (Anderson et al., Science 230: 144-149 (1985); Miller et al., J.Biol. Chem. 262: 9016 (1987)) expressed in appropriate host strains(2,5-DKG producers) such as Erwinia sp. These results have been achieveddespite 2,5-DKG reductase A having a low specificity constant for2,5-DKG. Since Corynebacterium does not naturally encounter 2,5-DKG, itis not surprising that this compound is a poor substrate for 2,5-DKGreductase A.

This low specificity constant for 2,5-DKG reductase A is in contrast toa second, homologous Corynebacterium 2,5-DKG reductase (2,5-DKGreductase B, also known as 2,5-DKG reductase I) that has a greaterspecificity constant for 2,5-DKG (Sonoyama and Kobayashi, J. Ferment.Technol. 65: 311-317 (1987)). In addition, both 2,5-DKG reductases arehomologous to several known aldose and keto-reductases that have greaterspecificity constants towards their known substrates. Such findingsindicate that the active site of 2,5-DKG reductase A is not optimallyconfigured for the catalytic conversion of 2,5-DKG to 2-KLG. Therefore,it appears that in order to optimize 2,5-DKG reductase A specificactivity in the single-fermentation process, amino acid substitutions bysite-directed mutagenesis must be made to the enzyme's active site.

In addition to improving an enzyme's kinetic parameters, site-directedmutagenesis can increase structural stability by amino acidsubstitutions, deletions, or insertions. The following are examples ofstructurally stabilizing mutations. The introduction of new disulfidebonds to create covalent crosslinks between different parts of a proteinhas been used to improve the thermal stability of bacteriophage T4lysozyme (Matsumura et al., Nature 342:291-293 (1989)), bacteriophage λrepressor (Sauer et al., Biochem. 25:5992-5998 (1986)), E. colidihydrofolate reductase (Villafranca et al., Biochem. 26:2182-2189(1987)), and subtilisin BPN' (Pantoliano et al., Biochem. 26:2077-2082(1987)). There is a computer program (Pabo et al., Biochem. 25:5987-5991(1986)) that permits efficient scanning of the crystallographicallydetermined three-dimensional structure of a protein to suggest thosesites where insertion of two cysteines might lead to disulfide bonds.Such bonds would not disrupt the larger-scale conformation, whilestabilizing the local conformation.

Amino acid substitutions of alanine for glycine in the α-helix have beenshown to increase the thermal stability of the bacteriophage λ repressor(Hecht et al., Proteins: Struct. Funct. Genet. 1:43-46 (1986)) and theneutral protease from Bacillus stearothermophilus (Imanaka et al.,Nature 324:695-697 (1986)). An increase in the melting temperature, Tm,for bacteriophage T4 lysozyme was accomplished by the two amino acidsubstitutions of proline for alanine and alanine for glycine (Matthewset al., Proc. Nat. Acad. Sci. USA 84:6663-6667 (1987)). Replacement ofamino acids in the hydrophobic core of a protein with aromatic residuessuch as tyrosine, especially at positions near preexisting clusters ofaromatic side chains, has been shown to promote thermal stability inkanamycin nucleotidyl transferase (Liao et al., Biochem. 83:576-580(1986)) and bacteriophage λ repressor (Hecht et al., Biochem.81:5685-5689 (1984)).

Transcriptional and translational control sequences in expressionvectors are the key elements required for the high level production ofproteins in bacteria. The E. coli Trp, bacteriophage λP_(L), E. coli lacUV5, and the Trp-lacUV5 fusion (Tac) promoters are among the mostfrequently used prokaryotic promoters (de Boer et al., Proc. Nat. Acad.Sci. USA 80: 21-25 (1983); Sambrook et al., Molecular Cloning, ColdSpring Harbor Press (1989); Remaut et al., Gene 15:81-93 (1981)). Thereis no way to determine whether a particular protein will be highlyexpressed upon induction of transcription from these promoters. Thetranslational efficiency of the message, mRNA stability, and theprotein's intrinsic stability are the major factors in high-levelexpression. Therefore, whenever a protein undergoes mutagenesis it isalways possible its expression level will be affected.

Site-directed mutagenesis, using synthetic DNA oligonucleotides havingthe desired sequence, permits substitution, deletion, or insertion ofselected nucleotides within a DNA sequence encoding a protein ofinterest. Recombinant DNA procedures are used to introduce the desiredmutation by substituting the synthetic sequence for the target sequence.Development of plasmids containing an origin of replication derived froma filamentous bacteriophage (Vieira and Messing, Methods in Enzymology153: 3-11 (1987)) permits cloning of fragments into single strandedforms of plasmids capable of autonomous replication. Use of suchplasmids eliminates the arduous task of subcloning DNA fragments fromplasmids to filamentous bacteriophage vectors. Kits for carrying outsite-directed mutagenesis are commercially available.

Mutants of 2,5-DKG reductase A having characteristics which vary fromthe native enzyme would be useful. In particular, mutants havingimproved catalytic activity, enhanced thermal stability, and increasedexpression levels would be useful to extend the commercial utility ofthe enzyme.

Unfortunately, unless proteins share regions of substantial sequence orstructural homology, it is not possible to generalize among proteins topredict, based on a beneficial mutation of one protein, precisely wherethe sequence encoding another protein should be changed to improve theperformance of that protein. Therefore, it is necessary to undertake ananalysis of the precise structural and functional features of theparticular protein to be altered. This determines which amino acids toalter to produce a desired result, such as increased catalytic activity,thermostability, or expression.

The present invention provides mutated forms of enzymatically activeprokaryotic 2,5-DKG reductase A. Analysis of the structure of 2,5-DKGreductase A to select alterations encoding the enzyme to enhancestability, expression, and/or activity of resulting mutants wasundertaken. Site-directed mutagenesis of the sequence encoding theenzyme was designed to produce the mutants.

SUMMARY OF THE INVENTION

The present invention provides mutants containing specific modificationsof 2,5-DKG reductase A, and materials and methods useful in producingthese proteins, as well as modified microorganisms and cell lines usefulin their production. Other aspects of the invention include theexpression constructs and products thereof for the modified 2,5-DKGreductases as well as cloning vectors containing the DNA encoding themodified 2,5-DKG reductases.

The DNA encoding the wild-type 2,5-DKG reductase A is modified usingsite-directed mutagenesis employing a single stranded form of plasmidwhich enables the generation of a change at a selected site within thecoding region of the 2,5-DKG reductase A. By this method, a change isintroduced into isolated DNA encoding 2,5-DKG reductase A which, uponexpression of the DNA, results in substitution of at least one aminoacid at a predetermined site in the 2,5-DKG reductase A. Also using thismethod, a change is introduced into isolated DNA encoding 2,5-DKGreductase A which, upon transcription of the DNA, results insubstitution of at least one nucleotide at a predetermined site in themRNA of the 2,5-DKG reductase A which allows increased expression.

The modified 2,5-DKG reductases and coding sequences of the inventionmay exhibit improved stability, expression, and/or catalytic activity,and may have varied Km and Vmax.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an expression vector for the 2,5-DKG reductase A gene;

FIG. 2 shows an expression vector for producing mutant forms of 2,5-DKGreductase A; and

FIG. 3 (SEQ. ID No. 1) shows schematically a proposed model for 2,5-DKGreductase A.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, "wild-type" 2,5-DKG reductase A refers to a proteinwhich is capable of catalyzing the conversion of 2,5-DKGstereoselectively to 2-KLG. The wild-type enzyme is the enzyme prior tothe modifications as described herein. The enzyme is obtained from theCorynebacterium sp. derived from ATCC strain No. 31090 as described inU.S. Pat. No. 5,008,193, incorporated herein by reference.

"Mutant" in relation to the "wild-type" 2,5-DKG reductase A, refers to aprotein having a related amino acid sequence which has enzymaticactivity substantially the same as the reference 2,5-DKG reductase A inthat the enzyme converts 2,5-DKG to 2-KLG. However, it contains one ormore amino acid substitutions, deletions, or insertions of amino acidresidues. These residues have been selected by using certain approachesto predict those regions of the protein that are most likely to containactive site residues. One approach involves using secondary structuralpredictions to assign 2,5 DKG reductase A to an eight-stranded α/βbarrel structure. A number of modifications are undertaken to modify thegene to encode mutants of the enzyme with improved characteristicscompared to the wild-type enzyme, for converting 2,5-DKGstereoselectively into 2-KLG.

It is well understood in the art that many of the compounds discussed inthe instant specification, such as proteins and the acidic derivativesof saccharides, may exist in a variety of ionization states dependingupon their surrounding media, if in solution, or out of the solutionsfrom which they are prepared if in solid form. The use of a term suchas, for example, gluconic acid, to designate such molecules is intendedto include all ionization states of the organic molecule referred to.Thus, for example, both "D-gluconic acid" and "D-gluconate" refer to thesame organic moiety, and are not intended to specify particularionization states. It is well known that D-gluconic acid can exist inunionized form, or may be available as, for example, the sodium,potassium, or other salt. The ionized or unionized form in which thecompound is pertinent to the disclosure will either be apparent from thecontext to one skilled in the art or will be irrelevant. Thus, the2,5-DKG reductase A protein itself and its various mutants may exist ina variety of ionization states depending on pH. All of these ionizationstates are encompassed by the terms "2,5-DKG reductase A" and "mutantform of 2,5-DKG reductase A."

"Expression vector" includes vectors which are capable of expressing DNAsequences contained therein where such sequences are operably linked toother sequences capable of effecting their expression. It is implied,although not explicitly stated, that expression vectors must bereplicable in the host organisms either as episomes or as an integralpart of a chromosomal DNA. Clearly, a lack of replication would renderthem effectively inoperable. In sum, "expression vector" is also given afunctional definition. Generally, expression vectors of utility in DNArecombinant techniques are often in the form of "plasmids". Plasmidsrefer to either circular double stranded DNA molecules or circularsingle stranded DNA molecules, containing an origin of replicationderived from a filamentous bacteriophage. These DNA molecules, in theirvector form, are not linked to the chromosomes. Other effective vectorscommonly used are phage and non-circular DNA. In the presentspecification, "plasmid" and "vector" are often used interchangeably.However, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which are, orsubsequently become, known.

"Recombinant host cells", "host cell", "cells", "cell cultures" and soforth are used interchangeably to designate individual cells, celllines, cell cultures, and harvested cells which have been or areintended to be transformed with the recombinant vectors of theinvention. The terms also include the progeny of the cells originallyreceiving the vector.

"Transformed" refers to any process for altering the DNA content of thehost. This includes in vitro transformation procedures such as calciumphosphate or DEAE-dextran-mediated transfection, electroporation,nuclear injection, phage infection, or such other means for effectingcontrolled DNA uptake as are known in the art.

The terms "amino acid" and "amino acids" refer to all naturallyoccurring L-α-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine. The amino acids are identifiedby either the single-letter or three-letter designations:

    ______________________________________                                        Asp  D       aspartic acid                                                                             Ile  I     isoleucine                                Thr  T       threonine   Leu  L     leucine                                   Ser  S       serine      Tyr  Y     tyrosine                                  Glu  E       glutamic acid                                                                             Phe  F     phenylalanine                             Pro  P       proline     His  H     histidine                                 Gly  G       glycine     Lys  K     lysine                                    Ala  A       alanine     Arg  R     arginine                                  Cys  C       cysteine    Trp  W     tryptophan                                Val  V       valine      Gln  Q     glutamine                                 Met  M       methionine  Asn  N     asparagine                                ______________________________________                                    

These amino acids may be classified according to the chemicalcomposition and properties of their side chains. They are broadlyclassified into two groups, charged and uncharged. Each of these groupsis divided into subgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

                  TABLE 1                                                         ______________________________________                                        Original Residue Conservative Substitutions                                   ______________________________________                                        Ala              ser                                                          Arg              lys                                                          Asn              gln; his                                                     Asp              glu                                                          Cys              ser; ala                                                     Gln              asn                                                          Glu              asp                                                          Gly              pro                                                          His              asn; gln                                                     Ile              leu; val                                                     Leu              ile; val                                                     Lys              arg; gln; glu                                                Met              leu; ile                                                     Phe              met; leu; tyr                                                Ser              thr                                                          Thr              ser                                                          Trp              tyr                                                          Tyr              trp; phe                                                     Val              ile; leu                                                     ______________________________________                                    

Substantial changes in function or stabilization are made by selectingsubstitutions that are less conservative than those in Table 1, i.e.,selecting residues that differ more significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site or (c)the bulk of the side chain. The substitutions which in general areexpected to produce the greatest changes will be those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteinyl or prolyl is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanyl, is substituted for (orby) one not having a side chain, e.g., glycyl.

General Methods

Most of the techniques which are used to transform cells, constructvectors, effect hybridization with a probe, carry out site-directedmutagenesis, and the like as well as the selection of mutants, arewidely practiced in the art. Most practitioners are familiar with thestandard resource materials which describe specific conditions andprocedures (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). However, foradditional guidance the following paragraphs are presented.

Expression of 2,5-DKG Reductase A

The complete functional gene is ligated into a suitable expressionvector containing a promoter and ribosome binding site operable in thehost cell into which the coding sequence will be transformed. In thecurrent state of the art, there are a number of promotion/controlsystems and suitable prokaryotic hosts available which are appropriateto the present invention. Similar hosts can be used both for cloning andfor expression since prokaryotes are, in general, preferred for cloningof DNA sequences. The method of 2-KLG production is most convenientlyassociated with such microbial systems. E. coli K12 strain 294 (ATCC No.31446) is particularly useful as a cloning host. Other microbial strainswhich may be used include E. coli strains such as E. coli B, E. coliX1776 (ATCC No. 31537) and E. coli DH-1 (ATCC No. 33489). Forexpression, the aforementioned strains, as well as E. coli W3110 (F-,λ-, prototrophic ATCC No. 27325), bacilli such as Bacillus subtilus, andother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species may be used. A particularlypreferred group of hosts includes those cultures which are capable ofconverting glucose or other commonly available metabolites to 2,5-DKG.Examples of such hosts are generally found among the genera Acetobacter,Gluconobacter, Acetomonas, and Erwinia. The taxonomy and nomenclature ofthese genera are such that the same or similar strains are sometimesgiven different names. For example, Acetobacter cerinus used in theexample below is also referred to as Gluconobacter cerinus. Examples ofparticular hosts include but are not limited to, Erwinia herbicola ATCCNo. 21998 (also considered an Acetomonas albosesamae in U.S Pat. No.3,998,697); Acetobacter (Gluconobacter) oxydans subspecies melanozenes,IFO 3292, 3293 ATCC No. 9937; Acetobacter (Gluconobacter) cerinus IFO3263 IFO 3266; Gluconobacter rubiginous, IFO 3244; Acetobacter fragumATCC No. 21409; Acetobacter (Acetomonas) suboxydans subspeciesindustrious ATCC No. 23776.

In general, plasmid expression or cloning vectors or conjugativeplasmids containing replication and control sequences which are derivedfrom species compatible with the host cell are used in connection withthese hosts. The vector ordinarily carries a replication origin as wellas marker genes which are capable of providing phenotypic selection intransformed cells. For example E. coli is typically transformed usingpBR322, a plasmid derived from an E. coli strain (Bolivar et al., Gene2:95-113 (1977)). pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides easy means for identifying transformedcells. For use in expression, the pBR322 plasmid, or other microbialplasmid must also contain, or be modified to contain, promoters whichcan be used by the microbial organism for expression of its ownproteins. Those promoters most commonly used in recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al., Nature 275: 617-624 (1978); Itakura etal., Science 198:1056-1063 (1977); Goeddel et al., Nature 281:544-548(1979)) and a tryptophan (trp) promoter system (Goeddel et al., NucleicAcids Res. 8:4057-4074 (1980); EPO Application No. 0036776). While theseare the most commonly used, other microbial promoters have beendiscovered and utilized. Details concerning their nucleotide sequenceshave been published, enabling a skilled worker to ligate themfunctionally in operable relationship to genes in transformationvectors. (Siebenlist et al., Cell 20:269-281 (1980)).

By suitable cleavage and ligation, DNA sequences encoding 2,5-DKGreductase A can be included in the aforementioned vectors prepared asoutlined above. Any unnecessary or inhibitory sequences may be deletedand the prokaryotic enzyme may then be purified; or the intact or brokencells used directly as catalysts. Alternatively, the host may be chosenso that once transformed it is capable of effecting the entireconversion of glucose or other suitable metabolite to the desired 2-KLGproduct.

Both the wild-type plasmid DNA and mutant plasmid DNA for 2,5-DKGreductase A is transfected into a host for enzyme expression. Therecombinant host cells are cultured under conditions favoring enzymeexpression. Usually selection pressure is supplied by the presence of anantibiotic. The resistance to the antibiotic is encoded by the vector.Culture under these conditions results in enzyme yields greater than thewild-type enzyme synthesis of the parent organism. This is the case,even if it is the parent organism that is transformed.

Vector Construction For Mutagenesis

Anderson et al have described the construction of plasmid ptrpl-35 inU.S. Pat. No. 5,008,193, incorporated herein by reference, that containsthe cloned DKG reductase A gene under the control of the E. coli trppromoter (FIG. 1). A derivative of this plasmid is constructed, with afew minor modifications to facilitate construction and characterizationof mutant forms of 2,5-DKG reductase A. These modifications aredescribed below. The final plasmid construct is called pSStac.DKGR.AAAand is shown in FIG. 2.

A) The structural gene for 2,5-DKG reductase A is mutated to includethree new restriction enzyme sites to facilitate further mutagenesisstudies. These three sites are "silent," i.e., the amino acid sequenceof the resulting DKGR A protein remains unchanged.

B) The promoter in pSStac.DKGR.AAA is the tac II promoter described byde Boer et al (Proc. Nat. Acad. Sci. USA 80:21-25 (1983)) instead of thetrp promoter found in ptrpl-35. This is a modified version of the trppromoter containing the binding site for lac repressor, allowing theexpression of the gene to be regulated in cells expressing the lacrepressor.

C) The plasmid is further modified to include the origin of replicationfrom the single stranded filamentous phage f1. The use of this DNAsequence in plasmids is well known in the art to produce a singlestranded form of the plasmid for sequencing and mutagenesis.

Site-Directed Mutagenesis

After the desired modifications are selected, the DNA sequence encodingthe 2,5-DKG reductase A is subjected to site-directed mutagenesis tosubstitute nucleotides encoding selected amino acids at thepredetermined positions within the sequence.

The preferred procedure for site-directed mutagenesis is performed bycloning the DNA sequence encoding the wild-type enzyme into arecombinant plasmid containing an origin of replication derived from asingle-stranded bacteriophage. Then an appropriate primer is used toconvert a residue at an identified position for example, to aconservative amino acid replacement. A synthetic oligonucleotide primercomplementary to the desired sequence, except in areas of limitedmismatching, is used as a primer in the synthesis of a strandcomplementary to the single-stranded wild-type 2,5-DKG reductase Asequence in the plasmid vector. The resulting double-stranded DNA istransformed into a host bacterium. Cultures of the transformed bacteriaare plated on agar plates, permitting colony formation from single cellswhich harbor the plasmid. Theoretically, 50% of the colonies willconsist of plasmid containing the mutant form; 50% will have theoriginal sequence. The colonies are hybridized with radiolabelledsynthetic primer under stringency conditions which permit hybridizationonly with the mutant plasmid which will form a perfect match with theprobe. Hybridizing colonies are then picked and cultured, and the mutantplasmid DNA is recovered.

Selection Of Sites For Mutagenesis Of Mutants For The Wild-Type 2,5-DKGReductase A Gene

Crucial to selection of sites for mutagenesis is prediction of asecondary and tertiary structure of the wild-type enzyme. The secondarystructural predictions are carried out in the following manner. First,the sequences of 2,5 DKG reductases A and B, and five other homologousenzymes (prostaglandin F synthase, bovine lens and rat lens aldosereductase, human liver aldehyde reductase, and ρ-crystallin from frogeye lens) are aligned to reveal a number of conserved residues. Second,the sequences are subjected to a number of structure predictionalgorithms (Chou and Fasman, Adv. Enzymol. 47:45-148 (1978); Garnier etal., J. Mol. Biol. 120:97-120 (1978); Wilmot and Thornton, J. Mol. Biol.203: 221-232 (1988); Karplus and Schulz, Naturwissenschaften 72: 212-214(1985); Eisenberg et al., Proc. Nat. Acad. Sci. USA 81:140-144 (1984);Rose and Roy, Proc. Nat. Acad. Sci. USA 77:4643-4647 (1980)) well knownin the art. These predictions are collated and compared to derive arough model of the enzyme's secondary structure as an eight-stranded α/βbarrel. This secondary structure prediction is consistent with therecently solved secondary structures of homologous enzymes having thefold of an eight-stranded α/β barrel (Rondeau et al., Nature 355:469-472(1992); Wilson et al., Science 257:81-84 (1992)).

The barrel structure is composed of two components. The first componentis a core of eight twisted parallel beta strands arranged closetogether, like staves, into a barrel. Surrounding this barrel structureis a second component of eight alpha helices that are joined to the betastrands through loops of various lengths. This eight-stranded α/β barrelstructure is called the triosephosphate isomerase (TIM) barrel from theenzyme for which this structure was first observed. The folding patternof the α/β barrel is found in 17 enzymes whose crystal structures areknown. In fact, approximately 10% of known enzyme structures are α/βbarrels (Farber and Petsko, TIBS: 228-234 (June 1990)). The 17 known α/βbarrel enzymes have a common α/β barrel core; substrate and cofactorspecificity comes from the variable loops joining the beta strands andalpha helices.

The proposed secondary structure model for 2,5-DKG reductase A, based ona consensus of secondary structure predictions on members of the aldosereductase family (see above), is shown schematically in FIG. 3 (SEQ. IDNO: 1), where beta strands are represented by arrows and the alphahelices are shown as cylinders. Regions of polypeptide chain connectingthe predicted elements of secondary structure are indicated as ofundefined structure. There are N and C terminal extensions of 34 and 17amino acids, respectively. Such extensions in the TIM-barrel enzymesoften form alpha helices that fold back over the top or bottom of thebarrel. Some subset of the eight loops at the C terminus of the betasheet (towards the top of FIG. 3 (SEQ. ID NO: 1), as well as theC-terminal "tail" (positions 262 to 278) are thought to comprise theactive site of the enzyme, as in the other TIM-barrel enzymes. Althoughonly a rough model, this structure greatly facilitates rationalengineering of the enzyme, by allowing the focus towards those residuesfound in proposed active site loops. It will be apparent that additionalresidues near to those in the proposed loops and "tail" may alsocomprise part of the active site.

Such information as to which amino acids comprise the active site of anenzyme can be gained from knowledge of the actual three dimensionalshape of the enzyme in question, as obtained from x-ray crystallographicor NMR studies. In the case of 2,5-DKG reductase, no such structuralinformation yet exists in the published literature. Therefore, analternate strategy in such a case would be using the model for 2,5-DKGreductase A as an α/β barrel discussed above, to limit the possiblesingle amino acid replacements to those residues found in proposedactive site loops.

By such an approach, the three surface loops that are the substratebinding site of 2,5-DKG reductase A are identified. These loops are atpositions 165-168, 187-198, and 224-234. A set of twelve 2,5-DKGreductase A mutants is made in these loops. This set comprises nearlyall possible point substitutions from the 2,5-DKG reductase B sequence.Many of these mutants, show major reductions in activity for converting2,5-DKG to 2-KLG, even when only minor or conservative changes are madein the amino acids. One of the mutants, with a substitution of argininefor glutamine at position 192 in the 2,5-DKG reductase A sequence, hasan improved ability to convert 2,5-DKG into 2-KLG. The construction ofthis mutant, named "Q192R" is described in Example 2.

The twelve mutants are expressed in Acetobacter cerinus and assayed forconversion of 2,5-DKG to 2-KLG at the crude lysate stage. Table 2 belowincludes a comparison of the activities of the twelve 2,5-DKG reductaseA mutants against the wild-type 2,5-DKG reductase A and 2,5-DKGreductase B. Assays in Table 2 are carried out as described in Table 3below. The increased activity of Q192R at the crude lysate stage,although somewhat obscured by the high levels of background reductaseactivity as seen in the pBR322 control lysates, is nonethelesssignificant and reproducible. The data for the pBR322, wild-type enzyme,and Q192R mutant in Table 2 are the average of three separate lysateassays. By assuming a simple additive contribution of the backgroundreductase activity in these lysates, these data show that the Q192Rmutant is twice as active as the wild-type enzyme against 2,5-DKG.Characterization of the kinetic constants of purified Q192R yields animproved Km and Vmax for this enzyme relative to wild type 2,5-DKGreductase A. See Example 5. Thus, Q192R shows improvement over thenatural enzyme both in specificity (Km) and in turnover rate (Vmax).

In a manner similar to that described above, the C-terminal "tail" isalso identified as part of the active site. A truncation mutant isdesigned that results in polypeptide termination before the last eightamino acid residues of 2,5-DKG reductase A. This mutant is found to bewell expressed, and the cofactor binding site is preserved, but, asshown in Table 2 below, it is absolutely inactive using 2,5-DKG as asubstrate. By this criteria the C-terminal "tail" is inferred tocomprise part of the binding pocket for 2,5-DKG.

                  TABLE 2                                                         ______________________________________                                                                  % Wild-type                                         Lysate Identity           on 2,5-DKG                                          ______________________________________                                        pBR322 (control)              0%                                              2,5-DKG Reductase A           100%                                            2,5-DKG Reductase B           >>>600%                                         (alanine for glycine                                                                          G191A mutant  1%                                              at position 191)                                                              (arginine for glutamine                                                                       Q192R mutant  200%                                            at position 192)                                                              (glycine deleted                                                                              G193 deleted  0%                                              at position 193)                                                                              mutant                                                        (arginine for lysine                                                                          K194R mutant  8%                                              at position 194)                                                              (serine for tyrosine                                                                          Y195S mutant  6%                                              at position 195)                                                              (tyrosine for alanine                                                                         A167Y mutant  0%                                              at position 167)                                                              (phenylalanine for tyrosine                                                                   Y168F mutant  2%                                              at position 168)                                                              (proline for glutamine                                                                        Q169P mutant  15%                                             at position 169)                                                              (leucine for lysine                                                                           K225L mutant  5%                                              at position 225)                                                              (serine for phenylalanine                                                                     F227S mutant  12%                                             at position 227)                                                              (threonine for valine                                                                         V228T mutant  23%                                             at position 228)                                                              (proline for valine                                                                           V229P mutant  22%                                             at position 229)                                                              (stop codon at position 271)                                                                  truncation mutant                                                                           0%                                                              (missing last eight                                                           amino acids)                                                  ______________________________________                                    

Without using the α/β barrel model as guidance, a random search of allpossible single amino acid replacements is necessary. This requires theconstruction and assay of 170 such enzymes, reflecting the differencesbetween the enzymes for possible recruitment of 2,5-DKG reductase B-likeactivity onto the 2,5-DKG reductase A framework.

Glycine residues in alpha helices that can accept the increased bulk ofthe substituted methyl group, are substituted with alanine residues tointroduce stabilization. Introduction of aromatic amino acid residuessuch as tyrosine, phenylalanine, and tryptophan near aromatic clusterswithin the enzyme are also within the scope of the invention. Theseadditional aromatic residues stabilize the enzyme at sites where theintroduction of such aromatic groups will not distort the overallconformation.

Mutations at particular sites in a protein can lead to enhancedexpression of that protein in bacteria. At the present time there is noway to predict which mutations lead to enhanced expression. However, itis known that the factors of translational efficiency, mRNA stability,and increased protein stability play a key role in high-levelexpression.

Many of the other possible point mutants are generated in clusters ofone to four closely spaced amino acid substitutions. Of the mutantswhich are stably folded, only those falling in the 165-168 loop, 187-198loop, 224-234 loop, and C-terminal "tail" (262-278) exhibit activitysignificantly different from the wild type enzyme. This is additionalconfirmation that these loop and tail regions comprise the enzyme activesite.

Any number of mutations proposed herein may be combined in a singlemutant. Obviously, a particular substitution at one location rules outreplacement with another amino acid at that same location in thatparticular mutant.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

EXAMPLE 1 Construction Of Plasmid pSStac.DKGR.AAA For Mutagenesis

An aliquot of plasmid ptrpl-35 was digested with EcoRI and HindIIIrestriction enzymes and the resulting 1690 base pair fragment purifiedby agarose gel electrophoresis. This fragment was then ligated intoEcoRI and HindIII digested vector M13 mp19. The resulting recombinantphage (called M13 mp19. DKGRA) was used to isolate a single strandedtemplate form of the phage for subsequent mutagenesis. The template wasmutagenized with three oligonucleotides to introduce three newrestriction enzyme cleavage sites to the 2,5-DKG reductase A gene. Thesesites are all `silent` in that although they introduce a new restrictioncleavage site to the DNA sequence, the amino acid sequence of theprotein coded for remains unchanged, due to degeneracy in the geneticcode. The three mutagenic oligonucleotides and the changes introducedare as follows: 1) oligonucleotide XbaA has sequence5'CGCGAAGCTGGCTCTAGATCAGGTCGAC 3' (SEQ ID NO: 2) and introduces a newXbaI site at amino acid position 98; 2) oligonucleotide ApaA hassequence 5' ATCGTGGGGGCCCCTCGGTCAGGGC 3' and introduces a new ApaI siteat amino acid position 188; and 3) oligonucleotide KpnA has sequence 5'GAGGTCGACTGAGGTACCCGAACACCCG 3' (SEQ ID NO: 4) and introduces a new KpnIsite immediately following the stop codon (TGA) after the final aminoacid. The mutagenesis reaction and conditions were essentially the sameas described in Example 2 for the construction of mutant Q192R. Afterthe mutagenesis reaction, positives plaques were identified byhybridization to the mutagenic oligonucleotide under stringentconditions, and the entire coding region of the 2,5-DKG reductase Afragment was sequenced to confirm the mutations.

The plasmid pSStac.DKGR.AAA was constructed as a three way ligation ofthe following fragments: 1) EcoRI to HindIII from the mutagenized phageM13 mp19.DKGRA as described above, this contains the coding gene for2,5-DKG reductase A; 2) the PstI to EcoRI fragment (850 base pairs) fromplasmid ptac6 (ptac6 is equivalent to plasmid ptrpl-35 but contains thetac promoter as described in de Boer et al. (Proc. Nat. Acad. Sci. USA80:21-25 (1983)) instead of the trp promoter found in ptrpl-35), and 3)the .sup.˜ 4,000 base pair vector fragment from HindIII to PstI ofplasmid p690. The p690 plasmid is a derivative of plasmid pBR322 withthe RsaI/DraI restriction fragment from the genome of bacteriophage f1(nucleotides 5489-5946), containing the single-stranded DNA origin ofreplication, inserted into the PvuII site.

The three fragments described above were isolated by agarose gelelectrophoresis, purified, and ligated in approximately equimolarratios, and used to transform competent E. coli cells. The resultingcolonies were analyzed by restriction mapping to identify the correctconstruct, called pSStac.DKGR.AAA (FIG. 2).

EXAMPLE 2 Site-Directed Mutagenesis Of The 2,5-DKG Reductase A Gene

A. Preparation of Template DNA For Mutagenesis

E. coli cells (strain XL1-Blue, Stratagene Corporation) bearing plasmidpSStac.DKGR.AAA were grown in LB media (Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, A.1 (1989)) toearly log phase, and infected with helper phage VCS-M13 (Stratagene).Infection with helper phage provides needed factors for the packing andsecretion of the single-stranded form of plasmid pSStac.DKGR.AAA. Theinfected cells were grown overnight with shaking at 37° C., and the nextday the cells were removed by centrifugation at 10,000 rpm for 10minutes in a Sorvall SM24 rotor. The supernatant containing the packagedplasmid was retained and the cell pellet discarded. The packaged plasmidwas precipitated by the addition of 1/4 volume of 2.5 M NaCl, 20% PEG(polyethylene glycol). After addition the mixture was stored at roomtemperature for 20 minutes, and then the precipitate was recovered bycentrifugation.

The precipitate was dissolved in 0.4 ml of TE buffer (10 mM tris, pH7.5, 1 mM EDTA) and further purified by several sequential extractionswith an equal volume of 50:50 chloroform:phenol. After each extractionthe aqueous (upper) phase was retained. The pure plasmid wasprecipitated with 2 volumes of ice-cold ethanol. The precipitate wasrecovered by centrifugation and dissolved in TE buffer. Theconcentration of the plasmid was estimated by measuring the opticalabsorbance at 260 nm using the conversion of 1 OD₂₆₀ =40 μg of singlestranded DNA per milliliter. The concentration of the plasmid wasadjusted to 1 μg per ml with TE.

B. Phosphorylation Of Oligonucleotide Primer

A synthetic oligonucleotide with the sequence

5' GCCCCTCGGTCGCGGCAAGTACG 3' (SEQ ID NO: 5) was synthesized andphosphorylated as follows: the oligonucleotide was diluted to aconcentration of 5.0 OD₂₆₀ units per ml. Then 2.5 μl of oligonucleotidewas combined with 3 μl 10x kinase buffer (1 M tris pH 8.0, 100 mM MgCl₂,70 mM dithiothreitol, 10 mM ATP), 25 μl water, and 2 units of T4polynucleotide kinase (New England Biolabs). The mixture was incubatedat 37° C. for 15 minutes, then the kinase enzyme was inactivated byheating to 70° C. for 10 minutes.

C. Mutagenesis Reaction

Six μl of kinased primer were combined with 1 μg of template DNA and 2.5μl of 10x RB buffer (70 mM tris, pH 7.5, 50 mM mercaptoethanol, 550 mMNaCl, and 1 mM EDTA) in a total volume of 10.5 μl. The primer wasannealed to the template by heating the mixture to 65° C. for fiveminutes, then slowly cooling to room temperature over a 30 minuteperiod.

To the annealing mixture was added 1.5 μl of 10x RB buffer, 1 μl of 10mM ATP, 1 μl of 10 mM DTT (dithiothreitol), and 1 μl T4 DNA ligase (NewEngland Biolabs). After 10 minutes, 1 μl of 1 M MgCl₂, 1 μl of 5mMdNTP's (an equimolar mixture of dATP, dCTP, dGTP, and dTTP) and 0.5 μlof Klenow (large fragment of DNA polymerase I, New England Biolabs) wereadded, and the mixture incubated at 15° C. overnight.

The following day, frozen competent E. coli MutL cells were transformedwith an aliquot of the reaction mixture, and plated onto agar platescontaining antibiotic selection (12.5 μg/ml tetracycline, 50 μg/mlampicillin). Colonies bearing mutant plasmids were initially identifiedby hybridization to the original mutagenic oligonucleotide understringent conditions (Wood et al, Proc. Nat. Acad. Sci. USA 82:1585-1588(1988)). Mutant plasmids were then prepared in a single-stranded form asin Section A and confirmed by direct DNA sequencing of the plasmid(United States Biochemical Corporation, Sequenase sequencing kit). Theresulting mutant Q192R 2,5-DKG reductase A, as shown in Example 5, hadimproved catalytic activity in comparison to the wild-type 2,5-DKGreductase A.

EXAMPLE 3 Expression Of Wild-Type 2,5-DKG Reductase A In AcetobacterCerinus

Plasmid DNA was introduced into Acetobacter cerinus (ATCC No. 39140) byelectroporation, as described (Wirth et al, Mol. Gen. Genet. 216(1):175-177 (March 1989)) using a Genepulser apparatus (BioradCorporation). Cells were grown to mid-log phase (OD₅₅₀ .sup.˜ 0.2-0.8)in 100 ml LB medium and recovered by centrifugation at 5,000 rpm in aSorvall SS-34 rotor for 5 minutes at 4° C. The cells were resuspended inone half volume of ice-cold electroporation buffer (300 mM sucrose, 7 mMsodium phosphate buffer, pH 7.0, and 1 mM MgCl₂), again pelleted bycentrifugation, and finally resuspended in 1/20th volume ofelectroporation buffer, and stored on ice until use. Plasmid DNA (0.1 to1.0 μg) was added to a 0.4 cm electroporation cuvette (BioradCorporation) which contained 0.8 ml of the prepared Acetobacter cells.The cells and DNA were mixed in the cuvette and cooled on ice for 10minutes prior to electroporation. The cells and DNA were given a singlepulse at 2500 mV using a 25 uF capacitor setting, and immediatelydiluted to 3.0 ml with fresh LB media. The diluted cells were thenallowed to recover with shaking at 30° C. for 2 hours. Aliquots (10-100μl ) of the transformed cells were plated on selective media (LB agarplates containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline) andthe plates were grown overnight at 30° C.

EXAMPLE 4 Purification Of The Mutant Q192R And The Wild-Type 2,5-DKGReductase A

Single colonies from transformed Acetobacter cerinus cells were grown in200 mls of 2 X YT media (Sambrook et al., Molecular cloning: ALaboratory Manual, Cold Spring Harbor Press, A.3 (1989)) containingantibiotics (12.5 μg/ml tetracycline and 50 μg/ml ampicillin) at 30° C.overnight. The cells were recovered by centrifugation (15 minutes at8000 rpm in a Sorvall GS3 rotor) and stored frozen. The cells were thenthawed in 1/5 volume of lysis buffer (50 mM tris, pH 8.0, 50 mMEDTA,0.1% Tween, 2 mg/ml lysozyme) and lysed for two hours on ice. The lysedcells were again centrifuged as before, and the supernatant containingthe crude cell extract retained.

The 2,5-DKG reductase A protein was purified from the crude cell extractby chromatography on DEAE cellulose. DEAE cellulose (Whatman DE-52brand) was pre-equilibrated with 25 mM tris, pH 7.0. A total of 5.0 mlof the gel was poured into a disposable plastic chromatography column,to which was applied the crude cell extract. After all of the extracthad been bound to the column, the column was washed with two columnvolumes of 25 mM tris pH 7.0, then one volume of 25 mM tris pH 7.0containing 0.3 M NaCl, and finally the 2,5-DKG reductase A protein waseluted with 25 mM tris pH 7.0 containing 0.6 M NaCl. The preparationswere assayed for protein concentration by the bicinchoninic acid method(Methods in Enzymology182:60-62 (1990)) and checked for purity by SDSpolyacrylamide gel electrophoresis.

EXAMPLE 5 Kinetic Characterization Of The Wild-Type And The Mutant Q192R2,5-DKG Reductase A

The preparations of wild-type and mutant Q192R 2,5-DKG reductase Aenzymes were characterized kinetically as to their ability to reduce thesubstrate 2,5-DKG to 2-KLG. Assays were done in 1 ml total volume of 50mM tris, pH 7.0, containing 0.2 mM NADPH, a constant amount of enzyme(15-20 mg) and amounts of substrate varying from 2 to 14 mM. The assayswere done at 25° C., and the rate of substrate reduction was measuredspectrophotometrically by measuring the loss of absorbance at 340 nmwavelength (which is indicative of the oxidation of the cofactor NADPHto NADP+).

The data were analyzed according to the well-known Michaelis equation todetermine the kinetic parameters Vmax and Km using the Enzfit softwarepackage (Biosoft, Cambridge, UK) on a Epson desktop computer. Thewild-type 2,5-DKG reductase A had a Vmax for the 2,5-DKG substrate of7.8 μmoles per minute per milligram of protein, while the Q192R mutanthad a Vmax of 14.0, a 1.8 fold improvement. The Km or Michaelis constantof the wild-type enzyme was 28 mM, while the Km of the Q192R mutant was21 mM for this substrate. This led to a specificity constant (kcat/Km)of 140 M⁻¹ s⁻¹ for the wild-type enzyme and a specificity constant of335 M⁻¹ s⁻¹ for the Q192R mutant, a 2.4 fold improvement.

EXAMPLE 6 A Mutant of 2,5-DKG Reductase A With Increased In-VivoExpression

A mutant form of 2,5-DKG reductase A was discovered which, althoughhaving activity equivalent to the wild-type enzyme, had increasedamounts of the protein accumulating in the Acetobacter expression host.This mutant, named "HS1", contains three amino acid changes: asparaginereplaces threonine at position two, threonine replaces serine atposition five, and serine replaces valine at position seven. Thesynthesis of this mutant was directed by a 37 base oligonucleotide withthe sequence 5' AATTCTATGAACGTTCCCACCATCAGCCTCAACGAC 3'(SEQ ID NO: 6).The steps in the mutagenesis reaction were essentially the same asoutlined for construction of the Q192R mutant.

Table 3 below shows results of assays of crude cell lysates ofAcetobacter cerinus bearing either: plasmid pBR322, a control plasmidthat contains no 2,5-DKG reductase A sequence, pSStac.DKGR.AAA, theplasmid expressing the wild-type gene, or pSStac.DKGR.AAA.HS1, whichcontains the HS1 mutations. Crude cell extracts were prepared asdescribed in the section on purification of 2,5-DKG reductase A andQ192R mutant protein. Results are shown for triplicate cell cultures.

                                      TABLE 3                                     __________________________________________________________________________                 assay values*                                                                             average                                                                            % wild-type                                     __________________________________________________________________________    pBR322       -0.031, -0.030, -0.041                                                                    -0.034                                                                              0%                                             pSStac.DKGR.AAA                                                                            -0.158, -0.192, -0.186                                                                    -0.178                                                                             100%                                            pSStac.DKGR.AAA.HS1                                                                        -0.207, -0,214, -0.217                                                                    -0.213                                                                             124%                                            __________________________________________________________________________     *values are the changes in absorbance at 340 nm per minute per 50 μl o     crude cell extract, at a substrate concentration of 10 mM 2,5DKG and 0.2      mM NADPH in 50 mM tris, pH 7.0, 25° C.                            

In these assays of crude cell lysates, it is necessary to account forbackground reductase from the Acetobacter cerinus cells themselves. Thisamount of activity is represented in the pBR322 cultures and issubtracted from the other values in order to calculate a "% wild-typeactivity."

Assays were done as before, in 1.0 ml total volume of 50 mM tris, pH7.0, containing 0.2 mM NADPH, a single fixed amount of 2,5-DKG (10 mM),and 50 μl of crude cell lysate.

Cell cultures bearing pSStac.DKGR.AAA.HS1 consistently show a 20-30%increase in expression levels over cell cultures containing thewild-type plasmid pSStac.DKGR.AAA. This increase in expression may bedue to changes in mRNA stability, level of translation of the message,protein stability, or some combination of these effects.

EXAMPLE 7 A Mutant of 2,5-DKG Reductase A with Increased TemperatureStability

A mutant form of 2,5-DKG reductase A is discovered which has increasedtemperature stability in the Acetobacter expression host. This mutantcontains two amino acid changes: alanine replaces glycine at position55, and alanine replaces glycine at position 57. The synthesis of thismutant is directed by a base oligonucleotide with the sequence 5'GAAACGAAGAAGCGGTCGCGGCCGCGATCGCG 3'(SEQ ID NO: 7). The steps in themutagenesis reaction are essentially the same as outlined for theconstruction of the Q192R mutant.

EXAMPLE 8 2,5-DKG Reductase A Mutants With Reduced Activity

Mutant forms of 2,5-DKG reductase A were discovered which showed majorreductions in activity for converting 2,5-DKG to 2-KLG. The steps in themutagenesis reactions were essentially the same as outlined forconstruction of the Q192R mutant. The following base oligonucleotidesdirected the synthesis of such mutants showing reduced activity in theAcetobacter expression host: with a substitution of alanine for glycineat position 191 to construct the G191A mutant, 5'GGGGCCGCTCGCCCAGGGCAAGT 3' (SEQ ID NO: 8); with a deletion of glycine atposition 193 to construct the G193 deleted mutant, 5'CCGCTCGGTCAGAAGTACGACCT 3' (SEQ ID NO: 9); with a substitution ofarginine for lysine at position 194 to construct the K194R mutant, 5'CGGTCAGGGCCGCTACGACCTCT 3' (SEQ ID NO: 10); with a substitution ofserine for tyrosine at position 195 to construct the Y195S mutant, 5'TCAGGGCAAGTCGGACCTCTTCG 3' (SEQ ID NO: 11); with a substitution oftyrosine for alanine at position 167 to construct the A167Y mutant, 5'GCTGCACCCCTACTACCAGCAGC 3' (SEQ ID NO: 12); with a substitution ofphenylalanine for tyrosine at position 168 to construct the Y168Fmutant, 5' GCACCCCGCCTTCCAGCAGCGCG 3' (SEQ ID NO: 13); with asubstitution of proline for glutamine at position 169 to construct theQ169P mutant, 5' CCCCGCCTACCCGCAGCGCGAGA 3' (SEQ ID NO: 14); with asubstitution of leucine for lysine at position 225 to construct theK225L mutant, 5' GCACCTGCAGCTCGGTTTCGTGG 3' (SEQ ID NO: 15); with asubstitution of serine for phenylalanine at position 227 to constructthe F227S mutant, 5' GCAGAAGGGTTCGGTGGTCTTCC 3' (SEQ ID NO: 16); with asubstitution of threonine for valine at position 228 to construct theV228T mutant, 5' GAAGGGTTTCACCGTCTTCCCGA 3' (SEQ ID NO: 17); with asubstitution of proline for valine at position 229 to construct theV229P mutant, 5' GGGTTTCGTGCCCTTCCCGAAGT 3' (SEQ ID NO: 18); and with astop codon at position 271 to construct the truncation mutant, 5'GGGTCGCGTGTGAGCACACCCCG 3' (SEQ ID NO: 19).

As will be apparent to those skilled in the art in which the inventionis addressed, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the present invention described above, are, therefore, to beconsidered in all respects as illustrative and not restrictive. Thescope of the present invention is as set forth in the appended claimsrather than being limited to the examples contained in the foregoingdescription.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 19                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 278 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetThrValProSerIleValLeuAsnAspGlyAsnSerIleProGln                              151015                                                                        LeuGlyTyrGlyValPheLysValProProAlaAspThrGlnArgAla                              202530                                                                        ValGluGluAlaLeuGluValGlyTyrArgHisIleAspThrAlaAla                              354045                                                                        IleTyrGlyAsnGluGluGlyValGlyAlaAlaIleAlaAlaSerGly                              505560                                                                        IleAlaArgAspAspLeuPheIleThrThrLysLeuTrpAsnAspArg                              65707580                                                                      HisAspGlyAspGluProAlaAlaAlaIleAlaGluSerLeuAlaLys                              859095                                                                        LeuAlaLeuAspGlnValAspLeuTyrLeuValHisTrpProThrPro                              100105110                                                                     AlaAlaAspAsnTyrValHisAlaTrpGluLysMetIleGluLeuArg                              115120125                                                                     AlaAlaGlyLeuThrArgSerIleGlyValSerAsnHisLeuValPro                              130135140                                                                     HisLeuGluArgIleValAlaAlaThrGlyValValProAlaValAsn                              145150155160                                                                  GlnIleGluLeuHisProAlaTyrGlnGlnArgGluIleThrAspTrp                              165170175                                                                     AlaAlaAlaHisAspValLysIleGluSerTrpGlyProLeuGlyGln                              180185190                                                                     GlyLysTyrAspLeuPheGlyAlaGluProValThrAlaAlaAlaAla                              195200205                                                                     AlaHisGlyLysThrProAlaGlnAlaValLeuArgTrpHisLeuGln                              210215220                                                                     LysGlyPheValValPheProLysSerValArgArgGluArgLeuGlu                              225230235240                                                                  GluAsnLeuAspValPheAspPheAspLeuThrAspThrGluIleAla                              245250255                                                                     AlaIleAspAlaMetAspProGlyAspGlySerGlyArgValSerAla                              260265270                                                                     HisProAspGluValAsp                                                            275                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGCGAAGCTGGCTCTAGATCAGGTCGAC28                                                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATCGTGGGGGCCCCTCGGTCAGGGC25                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAGGTCGACTGAGGTACCCGAACACCCG28                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCCCTCGGTCGCGGCAAGTACG23                                                     (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AATTCTATGAACGTTCCCACCATCAGCCTCAACGAC36                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAAACGAAGAAGCGGTCGCGGCCGCGATCGCG32                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGGGCCGCTCGCCCAGGGCAAGT23                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCGCTCGGTCAGAAGTACGACCT23                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CGGTCAGGGCCGCTACGACCTCT23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TCAGGGCAAGTCGGACCTCTTCG23                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCTGCACCCCTACTACCAGCAGC23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCACCCCGCCTTCCAGCAGCGCG23                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCCCGCCTACCCGCAGCGCGAGA23                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GCACCTGCAGCTCGGTTTCGTGG23                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GCAGAAGGGTTCGGTGGTCTTCC23                                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GAAGGGTTTCACCGTCTTCCCGA23                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GGGTTTCGTGCCCTTCCCGAAGT23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGGTCGCGTGTGAGCACACCCCG23                                                     __________________________________________________________________________

What is claimed is:
 1. A mutant form of a 2,5 diketo-gluconic acidreductase A, 2,5-DKG reductase A, enzyme having an ability to convert2,5-DKG into 2-KLG wherein said mutant form differs in amino acidsequence from (SEQ ID NO: 1) a wild-type form of a 2,5-DKG reductaseenzyme obtained from Corynebacterium sp. ATCC strain No. 31090, by:(A)the replacement of an amino acid residue selected from the groupconsisting of residues 2, 5, 7, 55, 57, 165, 166, 167, 168, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277 and 278 of said wild-typeenzyme or (B) the deletion of an amino acid residue selected from thegroup consisting of residues 2, 5, 7, 165, 166, 167, 168, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277 and 278 of said wild-type enzyme.2. The mutant according to claim 1 wherein said replacement is of anamino acid residue at a position selected from the group consisting of165, 166, 167, 168, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, and 278 and wherein said deletion is of an amino acid residue at aposition selected from the group consisting of 165, 166, 167, 168, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197,
 198. 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, 274, 275, 276, 277, and
 278. 3. The mutantaccording to claim 1 having an amino acid substitution in position 192.4. The mutant according to claim 3 having an arginine at position 192.5. The mutant form of 2,5-DKG reductase A of claim 1, wherein saidmutant form differs in amino acid sequence from a wild-type form of a2,5-DKG reductase enzyme obtained from Corynebacterium sp. ATCC strainNo. 31090, by the replacement of an amino acid residue selected from thegroup consisting of residues 2, 5, and 7 of said wild-type enzyme, andwherein promoter-directed expression, in Acetobacter strain ATCC 39140,of DNA encoding said mutant form causes said mutant form to beaccumulated to an even greater extent than that to which wild-type 2,5DKG reductase A form is accumulated when DNA encoding said wild-typeform is expressed directed by said promoter in said Acetobacter strain.6. The mutant according to claim 5 having an asparagine at position 2, athreonine at position 5, and a serine at position
 7. 7. The mutant formof 2,5-DKG reductase A of claim 1, wherein said mutant form has improvedtemperature stability, and wherein said mutant form differs in aminoacid sequence from a wild-type form of a 2,5-DKG reductase enzymeobtained from Corynebacterium sp. ATCC strain No. 31090, by thereplacement of an amino acid residue selected from the group consistingof residues 55, and 57 of said wild-type enzyme.
 8. The mutant accordingto claim 7 having an alanine at positions 55 and
 57. 9. The mutant formof 2,5-DKG reductase A of claim 5, having improved temperaturestability, wherein said mutant form additionally differs in amino acidsequence from a wild-type form of a 2,5-DKG reductase enzyme obtainedfrom Corynebacterium sp. ATCC strain No. 31090, by the replacement of anamino acid selected from the group consisting of residues 55 and 57 ofsaid wild-type enzyme.